The long-range goal of this research is to understand the molecular mechanisms associated with cell death in cells of the blood and vasculature. Necrotic cell death, or oncosis, can result in vascular cells from oxidant stress which may arise following activation of neutophils, uptake of low-density lipoproteins, or ischemia-reperfusion injury. Although a role for Ca2+ in necrotic cell death is well established, the molecular link(s) between a rise in cytosolic free Ca2+ concentration ([Ca2+]i) and subsequent cell lysis remains unknown. In recent studies, we have focussed on understanding the interaction of the potent marine toxin, maitotoxin (MTX), with endogenous cellular pathways leading to cell lysis. MTX, at picomolar concentrations, activates Ca2+-permeable, nonselective cation channels (CaNSC) leading directly to a sustained elevation of [Ca2+li. A secondary response to MTX is the formation of aqueous "pores" subsequent to the elevation of [Ca2+]i, which appear to grow in size, and allow passage of vital dyes into the cell. MTX-activated channels and pores are found in all mammalian cells examined to date including aortic endothelial cells, HEK293 cells, THP-1 monocytes, skin fibroblasts, and BW5147.3 lymphoma cells. The ubiquitous pattern of MTX-induced effects and the apparent high affinity suggests a mechanism of cytotoxicity that is specific and highly conserved throughout evolution. Indeed, the effects of MTX are indistinguishable from activation of P2Z/P2X7 purinergic receptors. In this regard, our most recent studies have shown that MTX and P2Z/P2X7 receptor stimulation activate distinct channels, but a common cytolytic pore. We have termed this ubiquitous pore, the cytolytic/oncotic pore, or COP. Using a combination of electrophysiological, biochemical, and molecular biological approaches, the specific aims of this proposal will test several hypotheses concerning the structure, function and regulation of COP. The specific aims are to determine 1) the molecular mechanism(s) by which CaNSC activity is linked to activation of COP, 2) the role of CaNSC activity and COP in activation of oncosis and/or apoptosis, and 3) the role of CaNSC and COP in oxidant-induced cell death. The proposed studies will increase our understanding of the molecular mechanisms associated with oncosis and apoptosis, provide a detailed analysis of the initial events involved in pore formation, and have important implications for cellular responses in vascular disease models.